Endothelial cell-conditioned medium enhanced astrocyte differentiation in cerebral organoids

Human brain organoids (hBOs) are self-organizing, three-dimensional structures that recapitulate aspects of early brain development, including astrogliogenesis. However, astrocyte differentiation does not occur until months into the differentiation process, and astrocytes continue to mature for years after that. Astrogliogenesis and astrocyte maturation could be further delayed by innate shortcomings of the model such as a lack of endothelial cells (ECs). Indeed, studies suggest that ECs or EC-derived factors promote astrogliogenesis and astrocyte maturation in vitro. Here, we exposed unguided hBOs to conditioned medium of immortalized human cerebral microvascular ECs (hCMEC/D3 cells) and determined their expression profile on the single-cell level at two time points. We found that EC-secreted proteins do not induce premature astrogliogenesis in hBOs but do affect hBO cellular composition and the maturation of radial glia, which are the precursors to astrocytes. These findings indicate the potential of EC-secreted factors to enhance astrocyte (precursor) maturation in hBOs and emphasize the importance of considering EC effects when aiming to accurately study developing astrocytes using hBOs. Overall design: Cerebral organoids were cultivated in a medium conditioned by endothelial cells. For scRNA-seq, they were harvested at 64 and 118 days in culture, along with their control counterparts cultivated in normal medium.